The objective of this project is to determine what chemical and ultrastructural modification of the outer membrane (OM) of Pseudomonas aeruginosa accompanies resistance to increasing concentrations of selected antibiotics, both in a model system and in clinical isolates. A model system for the development of resistance to the antibiotic polymyxin B has been developed in P. aeruginosa (Gilleland and Murray, J. Bacteriol. 125: 267-281, 1976). The OM of cells adapted to grow in the presence of 6000 units of polymyxin B per ml was shown to be ultrastructurally altered. Preliminary chemical analysis of cell envelopes from polymyxin-resistant isolates in the model system, from stable genetic mutants, and from clinical isolates by means of determiation of the total protein, carbohydrate, and 2-keto-3-deoxyoctonate content plus qualitative changes in protein content by slab polyacrylamide gel electrophoresis has shown the resistant cells have lost lipopolysaccharide and proteins identified as OM proteins. All the isolates will be examined by freeze-etching for ultrasturctural alteration of the OM and analyzed chemically as described above. The ability of polymyxin-resistant cells to bind the antibiotic will be determined using radioactively labeled polymyxin. Alteration in the lipid content of resistant versus sensitive cells will be studied, and the concentration of key receptor phospholipids for binding polymyxin will be determined in resistant versus sensitive cells employing thin-layer chromatography. Reconstitution studies in which phospholipid vesicles merge with the OM of resistant cells will be performed in order to restore polymyxin-sensitivity to the cells. Resistance to other selected antibiotics will be tested for in the polmyxin-resistant mutants. The use of both ultrasturctural and chemical analysis together should give a much clearer understanding of the role of the OM in mediating antibiotic resistance.